Most human leukemia are associated with chromosomal abnormalities resulting from genetic mutations or translocations that can create new hybrid genes capable of expressing mutated or fused proteins. The encoded abnormal fusion proteins are characterized by a joining region segment composed of a unique sequence of amino acids that is potentially immunogenic.
In chronic myelogenous leukemia (CML), the t(9;22) translocation results in a chimeric bcr-abl gene which encodes a 210 kD fusion protein. Two chimeric P210 bcr-abl proteins comprising products of either the b2a2 exon junction or the b3a2 exon junction can be alternatively expressed in CML cells. The junctional sequences represent unique tumor specific determinants, not only because they contain a joined set of amino acid sequences that are normally not expressed on the same protein, but also because at the exact fusion point, a codon for a new amino acid is present (1,2).
Similarly, a specific chromosomal translocation is present in acute promyelocytic leukemia (APL) cells which yield a 90-110 kD protein in which the product of the third exon of the RAR gene, located on chromosome 17, is fused to the amino terminal portion of a Zn-finger protein, PML from chromosome 15. Alternative breakpoints yield PML-RAR A and PML-RAR B that are observed in cells from 90% of APL patients (3,4). As with CML, both APL proteins contain an unique epitope consisting of the site of fusion in addition to a new amino acid.
Human T cell recognition of tumor associated antigens has been demonstrated for human melanoma antigens (5,6), as well as for a single point mutation in ras, resulting in a single amino acid change (7). Specific human CD4 T cell responses have been generated in vitro against the PML-RAR fusion protein found in APL cells (8), and in vivo, against B cell lymphoma immunoglobulin idiotypes (9).
The pairs of proteins found in CML and APL represent some of the most obvious targets for an immunological approach to the treatment of these leukemias and serve as a model for this approach in other neoplasms. Though the bcr-abl and PML-RAR proteins have an intracellular location, enzymatic degradation products of these fusion proteins could be presented on the cell surface as short peptides, 8-25 amino acids in length, within the cleft of HLA molecules, and potentially may be recognized by T cells. These peptides are derived by intracellular processing of exogenous and endogenous proteins as part of the antigen presentation pathway (10).
The amino acid motifs responsible for specific peptide-binding to HLA class I molecules have been determined for the common HLA class I types by the use of the analysis of acid-eluted naturally processed peptides and by use of cell lines defective in intracellular peptide loading and processing (11-13). More recently, a quantitative molecular radiobinding assay for the analysis of peptide binding to purified HLA class I molecules has been developed by Sette et al. (14).
In order to develop a vaccine strategy for APL and CML, the first two important questions are whether oncogenic fusion proteins contain suitable amino acid sequences and appropriate anchor motifs for binding to class I molecules and whether these breakpoint peptides can bind with sufficiently high affinity to the groove of HLA class I molecules; this activity is necessary to induce a leukemia specific T cell response. The ability of a series of synthetic peptides corresponding to the junctional sequences of bcr-abl and PML-RAR proteins to bind to purified human class I molecules is analyzed here. The rationale for this approach was twofold: 1) Breakpoint spanning peptides able to bind class I molecules could be potential candidate antigens for active immunotherapy against these leukemias. 2) Evidence that unique tumor specific breakpoint sequences are not presented in the context of HLA molecules would provide a molecular basis for immune non-responsiveness to abnormal intracellular fusion proteins in leukemic cells.
Cullis et al. (Leukemia (1994) 8:165-170), tested 18 peptides spanning the junctional sequences of the b2a2 and b3a2 protein for their ability to rescue the expression of the class I alleles in two human cell lines LBL 721.174 (T2) (HLA A2, B5) and BM 36.1 (HLA A1, B35). These cells are defective in intracellular peptide loading of class I molecules. None of the bcr-abl peptides enhanced HLA A2 or HLA B35 allele expression when compared with allele specific control peptides. The authors concluded that none of the CML peptides satisfies the known peptide-binding motifs for these alleles.
Gambacorti-Passerini, et al. was unable to demonstrate generation of a CD8/HLA class I restricted response to the fusion breakpoint peptides in acute promyelocytic leukemia (APL). (Blood (1994) 84(Suppl): p.618a, Abstract 2459).
Chen, et al. reported that immunization of mice with synthetic peptides corresponding to the BCR-ABL joining region elicited peptide-specific CD4.sup.+, class II major histocompatibility complex-restricted T cells. (Proc. Natl. Acad. Sci. USA (1992) 89: 1468-1472). Being human proteins, both BCR and ABL are foreign proteins to mice. Immunogenicity of a foreign peptide, even a foreign breakpoint peptide, does not directly address whether peptides spanning the joining region of two native proteins can stimulate an immunogenic response.
The data presented here is the first time that breakpoint peptides have been shown to be able to bind HLA class I molecules. In addition, such breakpoint peptides stimulated proliferation of human cytotoxic T-cells which demonstrated an ability to kill cells presenting the breakpoint peptide in the cleft of the appropriate HLA molecule.